Removal of protein from natural rubber latex articles

ABSTRACT

A method for lowering the extractable protein content in a natural rubber latex to below 30 parts per million (ppm) comprises the steps of treating by washing a rubber article, such as a rubber glove, with an aqueous solution containing a soluble silicate and, optionally, a protease. The methods claimed effectively reduce the allergenicity of the articles so treated.

TECHNICAL FIELD

This invention relates to the production of natural rubber latexarticles having a low protein content.

BACKGROUND ART

Articles made from natural rubber latex generally contain protein, andthis may give rise to a variety of undesirable effects in the finishedarticle, including allergic reactions in articles intended for use incontact with human skin, e.g. surgical gloves, medical catheters,household gloves and condoms.

The protein content in rubber latex articles is commonly expressed intwo different ways: as total extractable protein (EP) and as proteinantigen. The total extractable protein (EP) can be measured, e.g., bythe Lowry protein assay. The protein antigen expresses the allergenicityof the protein in the article; it may be measured, e.g., by the ELISAassay method.

A number of methods have been proposed to reduce the protein content orto alleviate the allergic reactions in articles made of natural rubberlatex. Thus, Novo Nordisk A/S in Research Disclosure, Mar. 10, 1992, No.335102 discloses that treatment of latex or latex goods with a proteaseis effective for changing the proteins to non-antigens and also forreducing the amount total protein in latex goods such as gloves.AU-A-44456/93 (Sumitomo Rubber Ind. & Kao Corp.) discloses a treatmentof natural rubber latex with a protease, an anionic surfactant and/or anon-ionic surfactant. AU-A-63069/94 and AU-A-63070/94 (both to SumitomoRubber Ind. & Kao Corp.) disclose a treatment of a natural rubber latexwith a protease and a surfactant, followed by the addition of, ananionic, amphoteric or non-ionic surfactant, oligomer or polymer, toimprove the mechanical stability.

SUMMARY OF THE INVENTION

Surprisingly, we have found that the extractable protein content of arubber latex article can be reduced by washing the article with asolution containing a soluble silicate. In a highly preferredembodiment, the solution also comprises a protease which serves tofurther reduce the extractable protein content and particularly to makethe remaining protein less antigenic. With a combination of silicate andprotease it is possible to reduce the protein antigen content to belowthe detection limit of the commonly used ELISA method.

Accordingly, the invention provides a method for producing a naturalrubber latex articles, comprising washing the article with a solutioncomprising a soluble silicate and optionally a protease.

Another aspect of the invention provides a composition comprising aprotease and a soluble silicate, in amounts which make the compositioneffective for use in lowering the extractable protein content of anarticle made of natural rubber latex.

DETAILED DESCRIPTION OF THE INVENTION

Natural Rubber Latex Article

The process of the invention is applicable to reducing the content ofextractable protein in any article made of natural rubber article,particularly latex-dipped products. Some specific examples are surgicalgloves, medical catheters, household gloves and condoms.

Soluble Silicate

The soluble silicate may be a crystalline alkaline silicate or acolloidal neutral silicate. It is preferably an alkali metal silicate,particularly a sodium silicate, preferably having a ratio of Na₂ O/SiO₂in the range from 2:1 to 1:3.75. Some specific examples are:

    ______________________________________                                        Chemical name  Empirical formula                                                                           Na.sub.2 O/SiO.sub.2  ratio                      ______________________________________                                        Sodium orthosilicate                                                                         Na.sub.4 SiO.sub.4                                                                          2:1                                              Sodium sesquisilicate                                                                        Na.sub.6 Si.sub.2 O.sub.7                                                                   1.5:1                                            Sodium metasilicate                                                                          Na.sub.2 SiO.sub.3                                                                          1:1                                              Colloidal neutral silicate                                                                   --            1:1.6 to 1:3.75                                  ______________________________________                                    

A suitable concentration of the soluble silicate is 0.2-10 g/l,preferably 1-5 g/l, in the solution used in the process of the inventionor 10-90% by weight of the composition of the invention.

Protease

Any type of protease known to be effective on latex extractable proteinor effective as a detergent additive can be used in the invention. Analkaline protease is preferred, e.g. an alkaline protease derived fromBacillus or Aspergillus, particularly a subtilisin. Some preferredproteases are Subtilisin 147 and Subtilisin 309 described in WO 89/06279and Subtilisin Carlsberg. Also, a mixture of two or more proteases canbe used, e.g. a mixture of 5 proteolytic components from Aspergillusoryzae described in WO 94/25580. Suitable protease preparations arecommercially available from Novo Nordisk A/S under the trade namesAlcalase®, Savinase®, Esperase® and Flavourzyme™. They are availableboth in liquid form and as non-dusting granulates.

The required concentration of the protease depends on the washingconditions (time, temperature etc.). For 5-15 minutes washing at 30-50°C., a suitable protease concentration corresponds to 0.5-100 KNPU/L,particularly 10-100 KNPU/L in the solution used in the process of theinvention or 0.2-2 KNPU/g of the composition of the invention. KNPU is aprotease activity unit, defined in U.S. Pat. No. 3,840,433.

Optional Ingredients

The removal of extractable protein may be enhanced by further adding asurfactant, preferably an amphoteric surfactant, and/or a second solubleelectrolyte (in addition to the soluble silicate).

However, the use of an amphoteric surfactant tends to make the rubberlatex articles more sticky and to increase foaming of the solution. Itmay therefore be preferred to avoid the use of a surfactant.

Also, other conventional additives may be included to enhance the effectof the protease, such as polyethylene glycol (PEG) and polymers like apolyacrylate or polyvinyl pyrrolidone.

Surfactant

If it is desired to use a surfactant, this may be amphoteric, anionic,non-ionic or a combination of these. It is particularly preferred to usean amphoteric surfactant or a combination of anionic and non-ionicsurfactant.

A suitable concentration of the surfactant is 0.2-10 g/l, particularly1-5 g/l in the solution used in the process of the invention or 3-30% byweight of the composition of the invention.

Amphoteric Surfactant

The amphoteric surfactant may be a propionate, e.g. tallowampho-poly-carboxy-propionate (e.g. Ampholak™ 7TY); animino-dipropionate, e.g. complex coco-imino-dipropionate (e.g. AmpholakYCE); an alkyl betaine, e.g. C-cetyl betaine having the formula (CH₃)₃N⁺ --CHR--COO (R=cetyl), available under the trade name Aquarex™ NS; analkylamido betaine; an imidazoline derivative, or an alkyl poly-aminocarboxylate (APAC).

A preferred amphoteric surfactant is a tallowampho-poly-carboxy-glycinate, particularly N-tallow alkylpoly-trimethylene poly-carboxymethyl sodium salts, available in aqueoussolution as Ampholak™ 7TX from Berol Nobel. It has the formula:

    R--(N--X)--(CH.sub.2).sub.3 --(N--X)--(CH.sub.2).sub.3 --(N--X)--(CH.sub.2).sub.3 --N--X

wherein R=C₁₆₋₁₈ and X=--CH₂ --COONa.

Another preferred amphoteric surfactant isoleo-ampho-poly-carboxy-glycinate, available in powder form as AmpholakX07-SD55 from Berol Nobel.

Anionic Surfactant

The anionic surfactant may be an α-olefin sulfonate (AOS), E.G. Lion™AOS, a linear alkylbenzene sulfonate (LAS), e.g. Miranil™, or an alkylsulphate, e.g. lauryl sulfate or Sipon™ LS from Henkel.

Non-ionic Surfactant

The non-ionic surfactant may be an alcohol ethoxylate, e.g. Elfapur™ LM75 S from Akzo Chemie or Dobange™ from Berol Nobel, or it may be a nonylphenol ethoxylate, e.g. Elmulwin™ W from Bayer.

Second Soluble Electrolyte

The second soluble electrolyte may be an alkali metal salt (particularlya sodium salt) such as a sulphate, a carbonate, a bicarbonate, achloride, a phosphate, a citrate or a combination of two or more ofthese.

A suitable concentration for the second soluble electrolyte is 1-10 g/l.

Washing Process

Suitable washing conditions are 1-60 minutes (particularly 5-30 minutes)washing at 20-70° C. (particularly 30-60° C.) and pH 10-12.5(particularly 10.5-12). A suitable weight ratio between the naturalrubber latex articles and the washing solution is in the range from 1:20to 1:5.

The washing may be performed with gentle mechanical stirring. Thewashing is typically preceded by a prewashing with water and followed byrinsing with water and drying. As mentioned above, there is generallyless foaming if the use of a surfactant is avoided, and this makes therinsing more effective.

Composition for Use in Protein Removal

The composition of the invention may be provided in powder, liquid orslurry form. A specific example of the formulation is a powderformulation (to be used at a dosage of about 17 g/l) consisting ofSavinase 8.0T (protease granulate with an activity of 8.0 KNPU/g) andsodium metasilicate at a weight ratio of 1:2 to 1:4.

EXAMPLES

In the Examples 1-4 and Comparative Example 1, natural rubber latexgloves were treated at the following conditions, unless otherwise noted:

Natural Rubber Latex Gloves

Weight: 9 g per glove

Extractable protein content: 600-1200 ppm (measured by the Lowrymethod).

The gloves used in each example were from the same batch.

Pre-wash

Temperature: ambient (22° C.)

Time: 15 minutes

Glove/water ratio: 1:10 w/w (e.g. 50 gloves of 9 g each washed with 4.5l of solution).

Stirring: manual

Liquid: Tap water

Washing

Temperature: 50° C.

Time: as noted

Glove/water ratio: 1:10 w/w

Stirring: manual

Liquid: distilled water with additives as given in each example

Rinsing

Temperature: ambient (22° C.)

Time: 5 minutes

Liquid: running tap water

Stirring: manual

Drying

Temperature: 70° C.

Time: 30 minutes

Extractable Protein Determination

Determination of extractable protein in ppm by Lowry assay.

Ingredients Used

The following ingredients were used in the examples. Concentrations aregiven in w/w %.

Protease: Savinase 16.0 L NR, protease activity 16.0 KNPU/g

Silicate: Sodium metasilicate

Amphoteric surfactant: Ampholak 7 TX

Non-ionic surfactant: alcohol ethoxylate (Elfapur or Dobange)

Anionic surfactant: alkyl sulfate (Sipon)

Sulphate: Sodium sulphate

Example 1

Natural rubber latex gloves were washed with the solutions shown below.The results are given below as ppm of extractable protein after 5 and 15minutes.

    ______________________________________                                        Test No.                                                                              1       2      3     4    5     6    7                                ______________________________________                                        Silicate                                                                              1       1      1     1    1     0.5  0.5                              Protease                                                                              0.1     0.1    0.1   0.1  0.1   0.1  0.1                              Amphoteric                                                                            --       --    0.5   0.5  1     1    0.5                              Non-ionic                                                                             --      --     --    --   --    --   0.5                              Sulphate                                                                              --      0.3    --    0.3  --    0.3  0.3                              5 min.  40      37     23    26   25    27   24                               15 min. 31      29     22    22   21    24   22                               ______________________________________                                    

It is seen that the extractable protein content can be brought down toabout 30 ppm after 15 minutes washing with silicate and protease, andthat it can be brought further down to about 22 ppm by addition ofamphoteric surfactant.

Example 2

Natural rubber latex gloves were washed with the solution shown below,with or without protease:

    ______________________________________                                               Silicate                                                                             0.3                                                                    Protease                                                                             0 or 0.1                                                               Anionic                                                                              0.5                                                                    Nonionic                                                                             0.1                                                                    Sulphate                                                                             0.3                                                             ______________________________________                                    

Total extractable protein was measured by Lowry, and the extractableprotein antigen was measured by ELISA. Results:

    ______________________________________                                               Without protease                                                                            With protease                                            Time, min.                                                                             Lowry, ppm                                                                              ELISA, ppm                                                                              Lowry, ppm                                                                            ELISA, ppm                               ______________________________________                                         5       38        0.6       33      0                                        10       35        0.35      30      0                                        15       24        0.65      25      0                                        ______________________________________                                    

The results show that silicate together with surfactant and sulphate waseffective in removing the extractable protein (Lowry), increasing withlonger washing time to a level below 30 ppm. The addition of a proteasewas very effective in removing the allergenicity of the remainingprotein to a level below the detection limit, and the protease alsoaccelerated the reduction of extractable protein.

Example 3

Repeated washing tests were carried out with 3 different formulations,as shown below, using gloves of different batches. The results are givenas average extractable protein content after 5, 10 and 15 minuteswashing.

    ______________________________________                                        Test No.   1             2      3                                             ______________________________________                                        Silicate   0.3           0.3    0.3                                           Protease   0.1           0.1    0.1                                           Anionic    0.5           0.6    0.5                                           Non-ionic  0.1           0.1    0.1                                           Sulphate   0.3           0.3    0.3                                           Repetitions                                                                              10            12     4                                             5 min.     42            38     37                                            10 min.    36            34     32                                            15 min.    34            29     28                                            ______________________________________                                    

The results show that the extractable protein content can be reduced toabout 30 ppm by 15 minutes washing with silicate and protease togetherwith surfactants and sulphate.

Comparative Example 1

For comparison, gloves were washed without silicate, in water with orwithout protease (0 or 0.1% Savinase). The total extractable protein wasmeasured by Lowry analysis, and the extractable protein antigen wasmeasured by ELISA. Results:

    ______________________________________                                               Without protease                                                                            With protease                                            Time min Lowry ppm ELISA ppm Lowry ppm                                                                             ELISA ppm                                ______________________________________                                        0        739       41.4      739     41.4                                     5        58        3.2       101     <0.6                                     15       57        2.2       77      <0.6                                     30       43        1.2       80      <0.6                                     ______________________________________                                    

The results show that a treatment with protease alone is effective inremoving protein antigens, but does not reduce the total amount ofextractable protein.

Example 4

Natural rubber latex gloves were washed for 15 minutes with thesolutions shown below. In this experiment, no pre-wash was used. Theresults are given as ppm of extractable protein. The control (untreatedgloves) contained 750 ppm of Lowry protein.

    ______________________________________                                                      Invention                                                                            Reference                                                ______________________________________                                        Silicate        1.0      --                                                   Protease        0.1        0.1                                                Amphoteric      0.5      --                                                   pH              12       12                                                   ppm protein     30       72                                                   ______________________________________                                    

The results show a significant reduction of the protease content by theuse of soluble silicate, compared to protease alone.

Example 5

In this experiment, natural rubber latex gloves were washed in a washingmachine, using the following procedure. The glove:water ratio was 1:10,and the time for 1 cycle wash was 1 hour.

    ______________________________________                                        Step   Operation       Time (min.)                                                                             Temperature                                  ______________________________________                                        1      Pre-wash        6         30                                           2      Drop bath       1         --                                           3      Main wash       15        50                                           4      Drop bath + spinning                                                                            1.5     --                                           5      Rinsing         5         30                                           6      Drop bath + spinning                                                                            1.5     --                                           7      Powder re-coating +                                                                           5         30                                                  spinning                                                               8      Drying          30        70                                           ______________________________________                                    

With each of the solutions shown below, the pH of the solution and theextractable protein (Lowry) after washing were measured. The control(untreated gloves) contained 750 ppm of Lowry protein. Results:

    ______________________________________                                                                              Lowry                                   Silicate    Amphoteric Protease pH    (ppm)                                   ______________________________________                                        Invention                                                                            0.5      --         --     10.5    22.5                                       0.5      0.5        --     10.6    14.5                                       0.5      --         0.1    10.5  18                                           0.5      0.5        0.1    10.6  17                                           1.0      0.5        0.1    11.8    10.5                                       0.3      --         0.1    10.2  28                                           0.2      0.2        0.1    10.2  18                                           0.1       0.05      0.1    10.2  25                                    Reference                                                                            --       --         --      9.5  74                                    ______________________________________                                    

The above results demonstrate the excellent performance of the solublesilicate. The protein content could be brought down to below 30 ppm bythe use of 0.5% silicate alone or by a combination of silicate withsurfactant and/or protease.

The stickiness of the gloves after the washing was comparedqualitatively. It was found that those treated with silicate togetherwith amphoteric surfactant were more sticky than those treated withsilicate alone, indicating that it may be preferable to avoid the use ofa surfactant.

What is claimed is:
 1. A method for lowering the extractable proteincontent of an article made of natural rubber latex, comprising washingthe article with an aqueous solution comprising a water-solublesilicate, under conditions in which said article, after said washing,contains about 30 or less ppm extractable protein.
 2. The method ofclaim 1 wherein the soluble silicate is an alkali metal salt of acrystalline alkaline silicate or colloidal neutral silicate.
 3. Themethod of claim 1, wherein the soluble silicate is a sodium salt of acrystalline alkaline silicate or colloidal neutral silicate having amolar ratio of Na₂ O/SiO₂ in the range from 2:1 to 1:3.75.
 4. The methodof the claim 1, wherein the soluble silicate is sodium metasilicate. 5.The method of claim 3, wherein the soluble silicate is a sodium salt ofa colloidal neutral silicate having a molar ratio of Na₂ O/SiO₂ in therange from 1:1.6 to 1:3.75.
 6. The method of claim 1 wherein the solublesilicate is present in said aqueous solution at a concentration of0.2-10 g/l.
 7. The method of claim 1, wherein the solution furthercomprises a protease.
 8. The method of claim 7 wherein the protease isan alkaline protease.
 9. The method of claim 7, wherein the protease isan alkaline Bacillus protease.
 10. The method of claim 7 wherein theprotease is present in said aqueous solution at a concentrationcorresponding to 0.5-100 protease activity units (KNPU)/L.
 11. Themethod of claim 1 wherein the solution further comprises a surfactant,wherein said surfactant is present in said solution at a concentrationof 0.2-10 g/l.
 12. The method of claim 1 wherein the solution furthercomprises an alkali metal salt selected from the group consisting of asulfate, a carbonate, a bicarbonate, a chloride, a phosphate, a citrate,or a combination of any of the foregoing, wherein said salt is presentin said solution at a concentration of 1-10 g/l.
 13. The method of claim1 wherein the solution has a pH in the range 10-12.5.
 14. The method ofclaim 1 wherein the washing is carried out at a temperature of 20-70° C.15. The method of claim 1 wherein the washing is carried out for aperiod of 1-60 minutes.
 16. The method of claim 1 wherein the naturalrubber latex article and the solution are present in a weight ratio of1:20 to 1:5.
 17. The method of claim 1, wherein said washing is precededby a pre-wash and followed by rinsing and drying.
 18. A method forlowering the extractable protein content of an article made of naturalrubber latex, comprising washing the article with an aqueous solutioncomprising (i) a water-soluble silicate and (ii) a protease, underconditions suitable for reducing allergenicity of said article.
 19. Themethod of claim 18 wherein said protease is Subtilisin 309.